There has long been an interest in the development of assay systems which can determine the presence or amount of specific substances in samples derived from biological specimens. Over the past two decades, immunoassays, which employ naturally occurring receptors directed to specific target substances, have provided valuable diagnostic tools for detecting substances of clinical significance. There are numerous immunoassays in the prior art in which one component of an immunological pair, e.g., an antigen or antibody, is detected or measured by using the complementary partner labelled with a tag which provides a detectible signal.
In one assay technique, known as a competitive binding technique, the substance to be detected competes with a labelled reagent of the same substance for a limited number of receptor sites. For example, for the detection of an unknown amount of a selected antigen in a liquid sample, a known amount of the labelled antigen is added to the sample and then contacted with receptor antibody specific for the antigen. The amount of labelled antigen which binds to the antibody is inversely proportional to the amount of the unknown antigen in the sample.
In another assay, known as a sandwich assay, receptor antibody is bound to a solid surface and the selected antigen in the sample binds to that antibody. A second labelled antibody capable of binding to the bound antigen is then reacted with the antigen to form an immobilized reaction product. The label in the reaction product is detected as an indication of the presence of the antigen in the sample.
For the detection or measurement of an antigen using a sandwich technique, antisera have been used for many years for both the labelled antibody and for the receptor antibody on the surface. More recently, monoclonal antibodies have been used in place of the antisera in such assay. In one such system, described in Wada, et al., Clin. Chem., 28(9):1986-1966 (1982), the receptor antibody was directed to one subunit of a particular antigen, hCG, while an enzyme-labelled monoclonal antibody was directed to another subunit. In this assay, the receptor antibody is immobilized on the inside of the test tube to which the sample was added.
Reaction on a solid surface can be relatively slow because the contact between the immobilized reagent and the analyte in the sample is limited. The assay time has been reduced by immobilizing the receptor antibody within a porous membrane, exposing the antibody molecules in a three-dimensional matrix. In many such systems the liquid sample containing the target antigen is drawn through the membrane into an underlying absorbent material. One such system, disclosed for use in a competitive binding assay, is U.S. Pat. No. 3,888,629. Other systems disclosed for use in competitive or sandwich assays include U.S. Pat. Nos. 4,246,339 and 4,366,241.
It is known to immobilize an antibody onto a membrane to bind an antigen and detect it, e.g. in a sandwich assay. The sample solutions and reagents flow through the membrane into an absorbent material on the other side of the membrane. One such system is described in U.S. Pat. No. 4,632,901. However, there is no suggestion that this type of system could be utilized for the detection of protein blots.
Protein blotting is a term used to describe the transfer of electrophoretically-resolved biological protein samples to an immobilizing matrix followed by a detection. Where the biological specimen is a protein, the blot is referred to as a Western blot. The general techniques for separations of the proteins and for blotting on the immobilized phase are well known (Techniques in Molecular Microbiology, J. Walker and W. Gaastre (Eds); G. Bers and D. Garfin, Bio Techniques, Vol. 3, No. 4, pp.276-288 (1985); Journal of Immunological Methods, 100 (1987) 281-282). As described, polyacrylamide gel electrophoresis is one suitable technique for this separation. Blotting matrices are available including nitrocellulose, diazobenzyloxymethyl (DBM) and diazophenylthioether (DPT)--modified cellulose paper and ion exchange papers (e.g. diethylaminoethyl (DEAE) cellulose). Nitrocellulose is a particularly effective blotting membrane. In a typical application, the proteins migrate through the gel under the influence of an electric field. The rate of migration is dependent on the charge, size and shape of the protein. The protein in the gel is then electrophoretically transferred to a support medium such as the nitrocellulose membrane.
After the blotting process, the protein on the membrane is detected in any of the aforementioned techniques. Commonly, antibody and various labelled materials such as an enzyme conjugate or a complex of colloidal gold with antibody has been used to detect the protein. It is known that the specificity of blotting may be difficult to control to avoid nonspecific binding to the membrane. Accordingly, a variety of blocking agents have been applied to the membrane prior to addition of the sample and labelled antibody. A common effective blocking agent is the surfactant polyoxyethylene sorbitan monolaurate surfactant (TWEEN-20). Such blocking agent is usually added at a low concentration, e.g. 0.05% polyoxyethylene sorbitan monolaurate surfactant (TWEEN-20).
Strips of nitrocellulose containing electrophoretically separated HIV-1 viral protein are commercially available for the detection of antibodies in an AIDS patient's serum. In such techniques, the strips are immersed into a solution of serum sample and labelled second antibody and mechanically agitated during incubation. Wash steps are normally required between incubations. Assay procedures of this type, termed overlays, typically require several hours. Thus, the procedure is labor and apparatus intensive.
Moreover, incubating such protein containing nitrocellulose strips with continuous agitation cause elution from the membrane of substantial portions of the blotted proteins. Another problem with this overlay approach is the protein blot is surrounded by an unstirred layer of solvent which restricts diffusion in mass transport across the liquid solid interface (Nernst law). This causes the antibody to be bound more quickly than it can be replenished by diffusion from the bulk solution. Thus, the observed rate constant is slower than expected and a slower reaction occurs.
A variety of label reagents have been used for anti-HIV immuno-blotting assays. Typically, such assays use a secondary antibody for visualizing antigen-bound primary antibodies. While enzymes have been used, gold labelled secondary antibodies have also been employed.